Folate Copolymer-Mediated Transfection of Cultured Cells

Abstract

Poly(ethylene glycol) of various sizes was used as a molecular spacer to separate the cell-targeting ligand, folate, from the surface of poly-L-lysine. The resulting ternary macromolecule (pLys-PEG-folate) was investigated in various formulations for its ability to transfect reporter plasmids into receptor-bearing HeLa and IGROV cell lines. Formulations were optimized with respect to DNA content, +/- charge ratio, and the size and amount of PEG substitution off the pLys backbone. Transfection activity was highest 48 h after sample introduction, and PEG 3400 was determined to be the most favorable spacer size tested. pLys-PEG-folate:DNA transfection was also found to be both concentration dependent and saturable; plus, it was blocked by the addition of excess-free folate, indicative of a specific mechanism of uptake. Transfection activity was virtually identical for complexes formed in 10% serum-supplemented media, deionized water, or Hepes buffer. And, cell viability remained greater than 85% at the highest concentrations of pLys-PEG-folate:DNA complexes tested (4.8 microg/mL pLys 331 000; 12 microg/mL DNA). Taken together, these observations provide evidence that pLys-PEG-folate:DNA complexes are taken up specifically by the folate endocytosis pathway, and that the intramolecular spatial distance of the ligand from the pLys backbone dramatically influences transfection.

Preparation and Characterization of Laminin-Derived Peptide AG73-Coated Liposomes as a Selective Gene Delivery Tool

“…Flow Cytometry Analysis 293T and 293T-Syn2 cells (6ϫ10 5 ) were treated with anti-syndecan-2 antibody (R&D Systems Inc., Minneapolis, U.S.A.) or isotype control antibody (Bechman Coulter, Inc., Brea, CA, U.S.A.) for 30 min at 4°C. Cells were subsequently treated with Alexa Fluor 488-conjugated secondary antibody (Invitrogen Japan K.K., Tokyo, Japan) for 30 min at 4°C, and then the fluorescence intensities were measured by flow cytometry.…”

“…Transfection of pDNA into Cells Using AG73-PEG Liposomes The 2 d before the experiments, 293T and 293T-Syn2 cells (1ϫ10 5 ) were seeded in a 48-well plate. The cells were treated with AG73-PEG liposomes (pDNA: 0.9 mg/ well) in serum-free medium for 4 h at 37°C.…”

“…Several different targeting moieties have been used in studies on cancer gene therapy, such as transferrin, folate, anisamide, RGD-peptide, and antibodies. [5][6][7][8][9][10] In this context, the present study focused on AG73 peptide, which is 12 amino acid synthetic peptide derived from the globular domain of the laminin a1 chain. AG73 peptide is as a ligand for syndecans, one of the major heparin sulfatecontaining transmembrane protein.…”

Potential use of folate-polyethylene glycol (PEG)-appended dendrimer (G3) conjugate with α-cyclodextrin as DNA carriers to tumor cells

“…Meanwhile, the previous reports demonstrated that an introduction of PEG as a spacer between FA and carriers increases FR-dependent gene transfer activity of liposomes 32 and poly-L-lysine. 33 To improve the FR-binding activity of Fol-a-CDE (G3), therefore, we prepared FolPaCs (G3), which have a PEG spacer between dendrimer and FA ( Figure 1), and examined FR-selective gene transfer activity of FolPaCs (G3, DSF 2, 5 or 7) in KB cells and A549 cells (Figure 3). In KB cells, Fol-PaC (G3, DSF 5) showed a higher gene transfer activity than a-CDE (G3) and Fol-PaCs (G3, DSF 2 and 7) (Figure 3a).…”

“…It should be noted that z-potential values of the pDNA complex with Fol-PaC (G3, DSF 5) at a charge ratio of 50 were close to neutral, compared with that of a-CDE (G3), because of an introduction of Fol-PEG moieties to dendrimer molecule. Leamon et al 33 reported that a neutral complex is desirable to incorporate into a ligand-targeted gene transfer carrier because it eliminates the opportunity for the non-specific adsorptive binding to non-target cells. Reddy et al 39,40 demonstrated that folate-linked carriers of diameters o150 nm are efficiently bound and internalized by FR-expressing cells.…”